Methods of modifying eukaryotic cells

ABSTRACT

A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification.

[0001] This application claims priority to U.S. patent Utilityapplication Ser. No. 09/732,234, filed Dec. 7, 2000, which claimspriority to U.S. Provisional Patent Application Serial No. 60/244,665,filed Oct. 31, 2000, each of which is incorporated by reference herein.Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application.

FIELD OF THE INVENTION

[0002] The field of this invention is a method for engineering andutilizing large DNA vectors to target, via homologous recombination, andmodify, in any desirable fashion, endogenous genes and chromosomal lociin eukaryotic cells. These large DNA targeting vectors for eukaryoticcells, termed LTVECs, are derived from fragments of cloned genomic DNAlarger than those typically used by other approaches intended to performhomologous targeting in eukaryotic cells. The field of the inventionfurther provides for a rapid and convenient method of detectingeukaryotic cells in which the LTVEC has correctly targeted and modifiedthe desired endogenous gene(s) or chromosomal locus(loci). The fieldalso encompasses the use of these cells to generate organisms bearingthe genetic modification, the organisms, themselves, and methods of usethereof.

INTRODUCTION

[0003] The use of LTVECs provides substantial advantages over currentmethods. For example, since these are derived from DNA fragments largerthan those currently used to generate targeting vectors, LTVECs can bemore rapidly and conveniently generated from available libraries oflarge genomic DNA fragments (such as BAC and PAC libraries) thantargeting vectors made using current technologies. In addition, largermodifications as well as modifications spanning larger genomic regionscan be more conveniently generated than using current technologies.Furthermore, the present invention takes advantage of long regions ofhomology to increase the targeting frequency of “hard to target” loci,and also diminishes the benefit, if any, of using isogenic DNA in thesetargeting vectors.

[0004] The present invention thus provides for a rapid, convenient, andstreamlined method for systematically modifying virtually all theendogenous genes and chromosomal loci of a given organism.

BACKGROUND OF THE INVENTION

[0005] Gene targeting by means of homologous recombination betweenhomologous exogenous DNA and endogenous chromosomal sequences has provento be an extremely valuable way to create deletions, insertions, designmutations, correct gene mutations, introduce transgenes, or make othergenetic modifications in mice. Current methods involve using standardtargeting vectors, with regions of homology to endogenous DNA typicallytotaling less than 10-20 kb, to introduce the desired geneticmodification into mouse embryonic stem (ES) cells, followed by theinjection of the altered ES cells into mouse embryos to transmit theseengineered genetic modifications into the mouse germline (Smithies etal., Nature, 317:230-234, 1985; Thomas et al., Cell, 51:503-512, 1987;Koller et al., Proc Natl Acad Sci USA, 86:8927-8931, 1989; Kuhn et al.,Science, 254:707-710, 1991; Thomas et al., Nature, 346:847-850, 1990;Schwartzberg et al., Science, 246:799-803, 1989; Doetschman et al.,Nature, 330:576-578, 1987; Thomson et al., Cell, 5:313-321, 1989;DeChiara et al., Nature, 345:78-80, 1990; U.S. Pat. No. 5,789,215,issued Aug. 4, 1998 in the name of GenPharm International) In thesecurrent methods, detecting the rare ES cells in which the standardtargeting vectors have correctly targeted and modified the desiredendogenous gene(s) or chromosomal locus(loci) requires sequenceinformation outside of the homologous targeting sequences containedwithin the targeting vector. Assays for successful targeting involvestandard Southern blotting or long PCR (Cheng, et al., Nature,369:684-5, 1994; Foord and Rose, PCR Methods Appl, 3:S149-61, 1994;Ponce and Micol, Nucleic Acids Res, 20:623, 1992; U.S. Pat. No.5,436,149 issued to Takara Shuzo Co., Ltd. ) from sequences outside thetargeting vector and spanning an entire homology arm (see Definitions);thus, because of size considerations that limit these methods, the sizeof the homology arms are restricted to less than 10-20 kb in total(Joyner, The Practical Approach Series, 293, 1999).

[0006] The ability to utilize targeting vectors with homology armslarger than those used in current methods would be extremely valuable.For example, such targeting vectors could be more rapidly andconveniently generated from available libraries containing large genomicinserts (e.g. BAC or PAC libraries) than targeting vectors made usingcurrent technologies, in which such genomic inserts have to beextensively characterized and trimmed prior to use. In addition, largermodifications as well as modifications spanning larger genomic regionscould be more conveniently generated and in fewer steps than usingcurrent technologies. Furthermore, the use of long regions of homologycould increase the targeting frequency of “hard to target” loci ineukaryotic cells, since the targeting of homologous recombination ineukaryotic cells appears to be related to the total homology containedwithin the targeting vector (Deng and Capecchi, Mol Cell Biol,12:3365-71, 1992). In addition, the increased targeting frequencyobtained using long homology arms could diminish any potential benefitthat can be derived from using isogenic DNA in these targeting vectors.

[0007] The problem of engineering precise modifications into very largegenomic fragments, such as those cloned in BAC libraries, has largelybeen solved through the use of homologous recombination in bacteria(Zhang, et al., Nat Genet, 20:123-8, 1998; Yang, et al., Nat Biotechnol,15:859-65, 1997; Angrand, et al., Nucleic Acids Res, 27:e16, 1999;Muyrers, et al., Nucleic Acids Res, 27:1555-7, 1999; Narayanan, et al.,Gene Ther, 6:442-7, 1999), allowing for the construction of vectorscontaining large regions of homology to eukaryotic endogenous genes orchromosomal loci. However, once made, these vectors have not beengenerally useful for modifying endogenous genes or chromosomal loci viahomologous recombination because of the difficulty in detecting rarecorrect targeting events when homology arms are larger than 10-20 kb(Joyner, The Practical Approach Series, 293, 1999). Consequently,vectors generated using bacterial homologous recombination from BACgenomic fragments must still be extensively trimmed prior to use astargeting vectors (Hill et al., Genomics, 64:111-3, 2000). Therefore,there is still a need for a rapid and convenient methodology that makespossible the use of targeting vectors containing large regions ofhomology so as to modify endogenous genes or chromosomal loci ineukaryotic cells.

[0008] In accordance with the present invention, Applicants providenovel methods that enables the use of targeting vectors containing largeregions of homology so as to modify endogenous genes or chromosomal lociin eukaryotic cells via homologous recombination. Such methods overcomethe above-described limitations of current technologies. In addition,the skilled artisan will readily recognize that the methods of theinvention are easily adapted for use with any genomic DNA of anyeukaryotic organism including, but not limited to, animals such asmouse, rat, other rodent, or human, as well as plants such as soy, cornand wheat.

SUMMARY OF THE INVENTION

[0009] In accordance with the present invention, Applicants havedeveloped a novel, rapid, streamlined, and efficient method for creatingand screening eukaryotic cells which contain modified endogenous genesor chromosomal loci. This novel methods combine, for the first time:

[0010] 1. Bacterial homologous recombination to precisely engineer adesired genetic modification within a large cloned genomic fragment,thereby creating a large targeting vector for use in eukaryotic cells(LTVECs);

[0011] 2. Direct introduction of these LTVECs into eukaryotic cells tomodify the endogenous chromosomal locus of interest in these cells; and

[0012] 3. An analysis to determine the rare eukaryotic cells in whichthe targeted allele has been modified as desired, involving an assay formodification of allele (MOA) of the parental -allele that does notrequire sequence information outside of the targeting sequence, such as,for example, quantitative PCR.

[0013] A preferred embodiment of the invention is a method forgenetically modifying an endogenous gene or chromosomal locus ineukaryotic cells, comprising: a) obtaining a large cloned genomicfragment containing a DNA sequence of interest; b) using bacterialhomologous recombination to genetically modify the large cloned genomicfragment of (a) to create a large targeting vector for use in theeukaryotic cells (LTVEC); c) introducing the LTVEC of (b) into theeukaryotic cells to modify the endogenous gene or chromosomal locus inthe cells; and d) using a quantitative assay to detect modification ofallele (MOA) in the eukaryotic cells of (c) to identify those eukaryoticcells in which the endogenous gene or chromosomal locus has beengenetically modified.

[0014] Another embodiment of the invention is a method wherein thegenetic modification to the endogenous gene or chromosomal locuscomprises deletion of a coding sequence, gene segment, or regulatoryelement; alteration of a coding sequence, gene segment, or regulatoryelement; insertion of a new coding sequence, gene segment, or regulatoryelement; creation of a conditional allele; or replacement of a codingsequence or gene segment from one species with an homologous ororthologous coding sequence from a different species.

[0015] An alternative embodiment of the invention is a method whereinthe alteration of a coding sequence, gene segment, or regulatory elementcomprises a substitution, addition, or fusion, wherein the fusioncomprises an epitope tag or bifunctional protein.

[0016] Yet another embodiment of the invention is a method wherein thequantitative assay comprises quantitative PCR, comparative genomichybridization, isothermic DNA amplification, or quantitativehybridization to an immobilized probe, wherein the quantitative PCRcomprises TaqMan® technology or quantitative PCR using molecularbeacons.

[0017] Another preferred embodiment of the invention is a method whereinthe eukaryotic cell is a mammalian embryonic stem cell and in particularwherein the embryonic stem cell is a mouse, rat, or other rodentembryonic stem cell.

[0018] Another preferred embodiment of the invention is a method whereinthe endogenous gene or chromosomal locus is a mammalian gene orchromosomal locus, preferably a human gene or chromosomal locus or amouse, rat, or other rodent gene or chromosomal locus.

[0019] An additional preferred embodiment is one in which the LTVEC iscapable of accommodating large DNA fragments greater than 20 kb, and inparticular large DNA fragments greater than 100 kb.

[0020] Another preferred embodiment is a genetically modified endogenousgene or chromosomal locus that is produced by the method of theinvention.

[0021] Yet another preferred embodiment is a genetically modifiedeukaryotic cell that is produced by the method of the invention.

[0022] A preferred embodiment of the invention is a non-human organismcontaining the genetically modified endogenous gene or chromosomal locusproduced by the method of the invention.

[0023] Also preferred in a non-human organism produced from thegenetically modified eukaryotic cells or embryonic stem cells producedby the method of the invention.

[0024] A preferred embodiment is a non-human organism containing agenetically modified endogenous gene or chromosomal locus, produced by amethod comprising the steps of: a) obtaining a large cloned genomicfragment containing a DNA sequence of interest; b) using bacterialhomologous recombination to genetically modify the large cloned genomicfragment of (a) to create a large targeting vector (LTVEC) for use inembryonic stem cells; c) introducing the LTVEC of (b) into the embryonicstem cells to modify the endogenous gene or chromosomal locus in thecells; d) using a quantitative assay to detect modification of allele(MOA) in the embryonic stem cells of (c) to identify those embryonicstem cells in which the endogenous gene or chromosomal locus has beengenetically modified; e) introducing the embryonic stem cell of (d) intoa blastocyst; and f) introducing the blastocyst of (e) into a surrogatemother for gestation.

[0025] An additional preferred embodiment of the invention is anon-human organism containing a genetically modified endogenous gene orchromosomal locus, produced by a method comprising the steps of: a)obtaining a large cloned genomic fragment containing a DNA sequence ofinterest; b) using bacterial homologous recombination to geneticallymodify the large cloned genomic fragment of (a) to create a largetargeting vector for use in eukaryotic cells (LTVEC); c) introducing theLTVEC of (b) into the eukaryotic cells to genetically modify theendogenous gene or chromosomal locus in the cells; d) using aquantitative assay to detect modification of allele (MOA) in theeukaryotic cells of (c) to identify those eukaryotic cells in which theendogenous gene or chromosomal locus has been genetically modified; e)removing the nucleus from the eukaryotic cell of (d); f) introducing thenucleus of (e) into an oocyte; and g) introducing the oocyte of (f) intoa surrogate mother for gestation.

[0026] Yet another preferred embodiment is a non-human organismcontaining a genetically modified endogenous gene or chromosomal locus,produced by a method comprising the steps of: a) obtaining a largecloned genomic fragment containing a DNA sequence of interest; b) usingbacterial homologous recombination to genetically modify the largecloned genomic fragment of (a) to create a large targeting vector foruse in eukaryotic cells (LTVEC); c) introducing the LTVEC of (b) intothe eukaryotic cells to genetically modify the endogenous gene orchromosomal locus in the cells; d) using a quantitative assay to detectmodification of allele (MOA) in the eukaryotic cells of (c) to identifythose eukaryotic cells in which the endogenous gene or chromosomal locushas been genetically modified; e) fusing the eukaryotic cell of (d) withanother eukaryotic cell; f) introducing the fused eukaryotic cell of (e)into a surrogate mother for gestation.

[0027] In preferred embodiments, the non-human organism is a mouse, rat,or other rodent; the blastocyst is a mouse, rat, or other rodentblastocyst; the oocyte is a mouse, rat, or other rodent oocyte; and thesurrogate mother is a mouse, rat, or other rodent.

[0028] Another preferred embodiment is one in which the embryonic stemcell is a mammalian embryonic stem cell, preferably a mouse, rat, orother rodent embryonic stem cell.

[0029] An additional preferred embodiment is the use of the geneticallymodified eukaryotic cells of the invention for the production of anon-human organism, and in particular, the use of the geneticallymodified embryonic stem cell of the invention for the production of anon-human organism.

[0030] A preferred embodiment of the invention is a method forgenetically modifying an endogenous gene or chromosomal locus ofinterest in mouse embryonic stem cells, comprising: a) obtaining a largecloned genomic fragment greater than 20 kb which contains a DNA sequenceof interest, wherein the large cloned DNA fragment is homologous to theendogenous gene or chromosomal locus; b) using bacterial homologousrecombination to genetically modify the large cloned genomic fragment of(a) to create a large targeting vector for use in the mouse embryonicstem cells, wherein the genetic modification is deletion of a codingsequence, gene segment, or regulatory element; c) introducing the largetargeting vector of (b) into the mouse embryonic stem cells to modifythe endogenous gene or chromosomal locus in the cells; and d) using aquantitative assay to detect modification of allele (MOA) in the mouseembryonic stem cells of (c) to identify those mouse embryonic stem cellsin which the endogenous gene or chromosomal locus has been geneticallymodified, wherein the quantitative assay is quantitative PCR. Alsopreferred is a genetically modified mouse embryonic stem cell producedby this method; a mouse containing a genetically modified endogenousgene or chromosomal locus produced by this method; and a mouse producedfrom the genetically modified mouse embryonic stem cell.

[0031] Another preferred embodiment is a mouse containing a geneticallymodified endogenous gene or chromosomal locus of interest, produced by amethod comprising the steps of: a) obtaining a large cloned genomicfragment greater than 20 kb which contains a DNA sequence of interest,wherein the large cloned DNA fragment is homologous to the endogenousgene or chromosomal locus; b) using bacterial homologous recombinationto genetically modify the large cloned genomic fragment of (a) to createa large targeting vector for use in the mouse embryonic stem cells,wherein the genetic modification is deletion of a coding sequence, genesegment, or regulatory element; c) introducing the large targetingvector of (b) into the mouse embryonic stem cells to modify theendogenous gene or chromosomal locus in the cells; and d) using aquantitative assay to detect modification of allele (MOA) in the mouseembryonic stem cells of (c) to identify those mouse embryonic stem cellsin which the endogenous gene or chromosomal locus has been geneticallymodified, wherein the quantitative assay is quantitative PCR; e)introducing the mouse embryonic stem cell of (d) into a blastocyst; andf) introducing the blastocyst of (e) into a surrogate mother forgestation.

[0032] Also preferred is the use of the genetically modified mouseembryonic stem cell described above for the production of a mouse.

[0033] One embodiment of the invention is a method of replacing, inwhole or in part, in a non-human eukaryotic cell, an endogenousimmunoglobulin variable region gene locus with an homologous ororthologous human gene locus comprising:

[0034] a) obtaining a large cloned genomic fragment containing, in wholeor in part, the homologous or orthologous human gene locus;

[0035] b) using bacterial homologous recombination to genetically modifythe cloned genomic fragment of (a) to create a large targeting vectorfor use in the eukaryotic cells (LTVEC);

[0036] c) introducing the LTVEC of (b) into the eukaryotic cells toreplace, in whole or in part, the endogenous immunoglobulin variablegene locus; and

[0037] d) using a quantitative assay to detect modification of allele(MOA) in the eukaryotic cells of (c) to identify those eukaryotic cellsin which the endogenous immunoglobulin variable region gene locus hasbeen replaced, in whole or in part, with the homologous or orthologoushuman gene locus.

[0038] Another embodiment is a method of replacing, in whole or in part,in a non-human eukaryotic cell, an endogenous immunoglobulin variableregion gene locus with an homologous or orthologous human gene locusfurther comprising the steps:

[0039] e) obtaining a large cloned genomic fragment containing a part ofthe homologous or orthologous human gene locus that differs from thefragment of (a);

[0040] f) using bacterial homologous recombination to genetically modifythe cloned genomic fragment of (e) to create a second LTVEC;

[0041] g) introducing the second LTVEC of (f) into the eukaryotic cellsidentified in step (d) to replace, in whole or in part, the endogenousimmunoglobulin variable gene locus; and

[0042] h) using a quantitative assay to detect modification of allele(MOA) in the eukaryotic cells of (g) to identify those eukaryotic cellsin which the endogenous immunoglobulin variable region gene locus hasbeen replaced, in whole or in part, with the homologous or orthologoushuman gene locus.

[0043] Another embodiment of the above method is a method wherein steps(e) through (h) are repeated until the endogenous immunoglobulinvariable region gene locus is replaced in whole with an homologous ororthologous human gene locus.

[0044] Another embodiment of the method is one in which theimmunoglobulin variable gene locus is a locus selected from the groupconsisting of:

[0045] a) a variable gene locus of the kappa light chain;

[0046] b) a variable gene locus of the lambda light chain; and

[0047] c) a variable gene locus of the heavy chain.

[0048] A preferred embodiment is a method wherein the quantitative assaycomprises quantitative PCR, FISH, comparative genomic hybridization,isothermic DNA amplification, or quantitative hybridization to animmobilized probe, and in particular wherein the quantitative PCRcomprises TaqMan® technology or quantitative PCR using molecularbeacons.

[0049] Yet another preferred embodiment is a method of replacing, inwhole or in part, in a mouse embryonic stem cell, an endogenousimmunoglobulin variable region gene locus with its homologous ororthologous human gene locus comprising:

[0050] a) obtaining a large cloned genomic fragment containing, in wholeor in part, the homologous or orthologous human gene locus;

[0051] b) using bacterial homologous recombination to genetically modifythe large cloned genomic fragment of (a) to create a large targetingvector for use in the embryonic stem cells;

[0052] c) introducing the large targeting vector of (b) into mouseembryonic stem cells to replace, in whole or in part, the endogenousimmunoglobulin variable gene locus in the cells; and

[0053] d) using a quantitative PCR assay to detect modification ofallele (MOA) in the mouse embryonic stem cells of (d) to identify thosemouse embryonic stem cells in which the endogenous variable gene locushas been replaced, in whole or in part, with the homologous ororthologous human gene locus.

[0054] In another embodiment, the method further comprises:

[0055] e) obtaining a large cloned genomic fragment containing a part ofthe homologous or orthologous human gene locus that differs from thefragment of (a);

[0056] f) using bacterial homologous recombination to genetically modifythe cloned genomic fragment of (e) to create a large targeting vectorfor use in the embryonic stem cells;

[0057] g) introducing the large targeting vector of (f) into the mouseembryonic stem cells identified in step (d) to replace, in whole or inpart, the endogenous immunoglobulin variable gene locus; and

[0058] h) using a quantitative assay to detect modification of allele(MOA) in the mouse embryonic stem cells of (g) to identify those mouseembryonic stem cells in which the endogenous immunoglobulin variableregion gene locus has been replaced, in whole or in part, with thehomologous or orthologous human gene locus.

[0059] Still another preferred embodiment is a method of wherein steps(e) through (h) above are repeated until the endogenous immunoglobulinvariable region gene locus is replaced in whole with an homologous ororthologous human gene locus.

[0060] Also preferred is a method wherein the immunoglobulin variablegene locus comprises a locus selected from the group consisting of

[0061] a) a variable gene locus of the kappa light chain;

[0062] b) a variable gene locus of the lambda light chain; and

[0063] c) a variable gene locus of the heavy chain.

[0064] Another preferred embodiment is a genetically modifiedimmunoglobulin variable region gene locus produced by the methodsdescribed above; a genetically modified eukaryotic cell comprising agenetically modified immunoglobulin variable region gene locus producedby the methods described above; a non-human organism comprising agenetically modified immunoglobulin variable region gene locus producedby the methods described above; and a mouse embryonic stem cellcontaining a genetically modified immunoglobulin variable region genelocus produced by the methods described above.

[0065] Also preferred is an embryonic stem cell wherein the mouse heavychain variable region locus is replaced, in whole or in part, with ahuman heavy chain variable gene locus; an embryonic stem cell of claimwherein the mouse kappa light chain variable region locus is replaced,in whole or in part, with a human kappa light chain variable regionlocus; an embryonic stem cell wherein the mouse lambda light chainvariable region locus is replaced, in whole or in part, with a humanlambda light chain variable region locus; and an embryonic stem cellwherein the heavy and light chain variable region gene loci arereplaced, in whole, with their human homologs or orthologs.

[0066] Another preferred embodiment is a mouse produced from theembryonic stem cells described above.

[0067] Yet another preferred embodiment is an antibody comprising ahuman variable region encoded by the genetically modified variable genelocus of described above; an antibody further comprising a non-humanconstant region; and an antibody further comprising a human constantregion.

[0068] Also preferred is a transgenic mouse having a genome comprisingentirely human heavy and light chain variable region loci operablylinked to entirely endogenous mouse constant region loci such that themouse produces a serum containing an antibody comprising a humanvariable region and a mouse constant region in response to antigenicstimulation; a transgenic mouse having a genome comprising human heavyand/or light chain variable region loci operably linked to endogenousmouse constant region loci such that the mouse produces a serumcontaining an antibody comprising a human variable region and a mouseconstant region in response to antigenic stimulation; a transgenic mousecontaining an endogenous variable region locus that has been replacedwith an homologous or orthologous human variable locus, such mouse beingproduced by a method comprising:

[0069] a) obtaining one or more large cloned genomic fragmentscontaining the entire homologous or orthologous human variable regionlocus;

[0070] b) using bacterial homologous recombination to genetically modifythe cloned genomic fragment(s) of (a) to create large targetingvector(s) for use in mouse embryonic stem cells;

[0071] c) introducing the large targeting vector(s) of (b) into mouseembryonic stem cells to replace the entire endogenous variable regionlocus in the cells; and

[0072] d) using a quantitative PCR assay to detect modification ofallele (MOA) in the mouse embryonic stem cells of (c) to identify thosemouse embryonic stem cells in which the entire endogenous variableregion locus has been replaced with the homologous or orthologous humanvariable region locus;

[0073] e) introducing the mouse embryonic stem cell of (d) into ablastocyst; and

[0074] f) introducing the blastocyst of (e) into a surrogate mother forgestation.

[0075] Another preferred embodiment is a transgenic mouse describedabove wherein the immunoglobulin variable region gene locus comprisesone or more loci selected from the group consisting of:

[0076] a) a variable gene locus of the kappa light chain;

[0077] b) a variable gene locus of the lambda light chain; and

[0078] c) a variable gene locus of the heavy chain.

[0079] Also preferred are the methods described above wherein the mouseembryonic stem cell is derived from a transgenic mouse produced by themethods.

[0080] Still yet another preferred embodiment of the invention is amethod of making a human antibody comprising:

[0081] a) exposing the mouse described above to antigenic stimulation,such that the mouse produces an antibody against the antigen;

[0082] b) isolating the DNA encoding the variable regions of the heavyand light chains of the antibody;

[0083] c) operably linking the DNA encoding the variable regions of (b)to DNA encoding the human heavy and light chain constant regions in acell capable of expressing active antibodies;

[0084] d) growing the cell under such conditions as to express the humanantibody; and

[0085] e) recovering the antibody.

[0086] In another preferred embodiment, the cell described above is aCHO cell.

[0087] Also preferred is a method of wherein the DNA of step (b)described above is isolated from a hybridoma created from the spleen ofthe mouse exposed to antigenic stimulation in step (a) described above.

[0088] Also preferred is the method described above wherein the DNA isisolated by PCR.

BRIEF DESCRIPTION OF THE FIGURES

[0089]FIG. 1: Schematic diagram of the generation of a typical LTVECusing bacterial homologous recombination.

[0090] (hb1=homology box 1; hb2=homology box 2; RE=restriction enzymesite).

[0091]FIG. 2: Schematic diagram of donor fragment and LTVEC for mouseOCR10.

[0092] (hb1=homology box 1; lacZ=9-galactosidase ORF; SV40 polyA=a DNAfragment derived from Simian Virus 40, containing a polyadenylation siteand signal; PGKp=mouse phosphoglycerate kinase (PGK) promoter; EM7=abacterial promoter; neo =neomycin phosphotransferase; PGK polyA=3′untranslated region derived from the PGK gene and containing apolyadenylation site and signal; hb2=homology box 2)

[0093] FIGS. 3A-3D: Sequence of the mouse OCR10 cDNA, homology box 1(hb1), homology box 2 (hb2), and TaqMan® probes and primers used in aquantitative PCR assay to detect modification of allele (MOA) in EScells targeted using the mOCR10 LTVEC.

[0094] hb1: base pairs 1 to 211

[0095] hb2: base pairs 1586 to 1801

[0096] TaqMan® probe and corresponding PCR primer set derived frommOCR10 exon 3:

[0097] TaqMan® probe: nucleotides 413 to 439—upper strand

[0098] Primer ex3-5′: nucleotides 390 to 410—upper strand

[0099] Primer ex3-3′: nucleotides 445 to 461—lower strand

[0100] TaqMan® probe and corresponding PCR primer set derived frommOCR10 exon 4:

[0101] TaqMan® probe: nucleotides 608 to 639—upper strand

[0102] Primer ex4-5′: nucleotides 586 to 605—upper strand

[0103] Primer ex4-3′: nucleotides 642 to 662—lower strand

[0104] FIGS. 4A-4D: Schematic diagram of the two LTVECs constructed toreplace the mouse VDJ region with human VDJ region.

[0105]FIG. 4A: Large insert (BAC) clones spanning the entire VDJ regionof the human heavy chain locus are isolated.

[0106]FIG. 4B: In this example, large insert (BAC) clones are isolatedfrom the ends of the mouse VDJ region as a source of homology arms whichare used to direct integration via homologous recombination of the humanVDJ sequences in a two step process.

[0107] FIGS. 4C-4D: In the first step, LTVEC1 (FIG. 4D) is constructedby bacterial homologous recombination in E. coli. LTVEC1 contains, inorder: a large mouse homology arm derived from the region upstream fromthe mouse DJ region, but whose absolute endpoints are not important; acassette encoding a selectable marker functional in ES cells(PGK-neomycinR in this example); a loxP site; a large human insertspanning from several V gene segments through the entire DJ region; anda mouse homology arm containing the region immediately adjacent to, butnot including, the mouse J segments. In the second step, LTVEC2 (FIG.4C) is constructed by bacterial homologous recombination in E. coli.LTVEC2 contains, in order: a large mouse homology arm containing theregion adjacent to the most distal mouse V gene segment, but notcontaining any mouse V gene segments; a large insert containing a largenumber of distal human V gene segments; a mutant loxP site called lox511in the orientation opposite to that of the wild type loxP sites inLTVEC2 and LTVEC1 (this site will not recombine with wild type loxPsites but will readily recombine with other lox511 sites); a wild typeloxp site; a second selectable marker (PGK-hygromycinR in this example);and a mouse homology arm derived from the V region, but whose absoluteendpoints are not important.

DEFINITIONS

[0108] A “targeting vector” is a DNA construct that contains sequences“homologous” to endogenous chromosomal nucleic acid sequences flanking adesired genetic modification(s). The flanking homology sequences,referred to as “homology arms”, direct the targeting vector to aspecific chromosomal location within the genome by virtue of thehomology that exists between the homology arms and the correspondingendogenous sequence and introduce the desired genetic modification by aprocess referred to as “homologous recombination”.

[0109] “Homologous” means two or more nucleic acid sequences that areeither identical or similar enough that they are able to hybridize toeach other or undergo intermolecular exchange.

[0110] “Gene targeting” is the modification of an endogenous chromosomallocus by the insertion into, deletion of, or replacement of theendogenous sequence via homologous recombination using a targetingvector.

[0111] A “gene knockout” is a genetic modification resulting from thedisruption of the genetic information encoded in a chromosomal locus.

[0112] A “gene knockin” is a genetic modification resulting from thereplacement of the genetic information encoded in a chromosomal locuswith a different DNA sequence.

[0113] A “knockout organism” is an organism in which a significantproportion of the organism's cells harbor a gene knockout.

[0114] A “knockin organism” is an organism in which a significantproportion of the organism's cells harbor a gene knockin.

[0115] A “marker ” or a “selectable marker” is a selection marker thatallows for the isolation of rare transfected cells expressing the markerfrom the majority of treated cells in the population. Such marker'sgene's include, but are not limited to, neomycin phosphotransferase andhygromycin B phosphotransferase, or fluorescing proteins such as GFP.

[0116] An “ES cell” is an embryonic stem cell. This cell is usuallyderived from the inner cell mass of a blastocyst-stage embryo.

[0117] An “ES cell clone” is a subpopulation of cells derived from asingle cell of the ES cell population following introduction of DNA andsubsequent selection.

[0118] A “flanking DNA” is a segment of DNA that is collinear with andadjacent to a particular point of reference.

[0119] “LTVECs” are large targeting vectors for eukaryotic cells thatare derived from fragments of cloned genomic DNA larger than thosetypically used by other approaches intended to perform homologoustargeting in eukaryotic cells.

[0120] A “non-human organism” is an organism that is not normallyaccepted by the public as being human.

[0121] “Modification of allele” (MOA) refers to the modification of theexact DNA sequence of one allele of a gene(s) or chromosomal locus(loci) in a genome. This modification of allele (MOA) includes, but isnot limited to, deletions, substitutions, or insertions of as little asa single nucleotide or deletions of many kilobases spanning a gene(s) orchromosomal locus (loci) of interest, as well as any and all possiblemodifications between these two extremes.

[0122] “Orthologous” sequence refers to a sequence from one species thatis the functional equivalent of that sequence in another species.

[0123] The description and examples presented infra are provided toillustrate the subject invention. One of skill in the art will recognizethat these examples are provided by way of illustration only and are notincluded for the purpose of limiting the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0124] Applicants have developed a novel, rapid, streamlined, andefficient method for creating and screening eukaryotic cells whichcontain modified endogenous genes or chromosomal loci. In these cells,the modification may be gene(s) knockouts, knockins, point mutations, orlarge genomic insertions or deletions or other modifications. By way ofnon-limiting example, these cells may be embryonic stem cells which areuseful for creating knockout or knockin organisms and in particular,knockout or knockin mice, for the purpose of determining the function ofthe gene(s) that have been altered, deleted and/or inserted.

[0125] The novel methods described herein combine, for the first time:

[0126] 1. Bacterial homologous recombination to precisely engineer adesired genetic modification within a large cloned genomic DNA fragment,thereby creating a large targeting vector for use in eukaryotic cells(LTVECs);

[0127] 2. Direct introduction of these LTVECs into eukaryotic cells tomodify the corresponding endogenous gene(s) or chromosomal locus(loci)of interest in these cells; and

[0128] 3. An analysis to determine the rare eukaryotic cells in whichthe targeted allele has been modified as desired, involving aquantitative assay for modification of allele (MOA) of the parentalallele.

[0129] It should be emphasized that previous methods to detectsuccessful homologous recombination in eukaryotic cells cannot beutilized in conjunction with the LTVECs of Applicants' invention becauseof the long homology arms present in the LTVECs. Utilizing a LTVEC todeliberately modify endogenous genes or chromosomal loci in eukaryoticcells via homologous recombination is made possible by the novelapplication of an assay to determine the rare eukaryotic cells in whichthe targeted allele has been modified as desired, such assay involving aquantitative assay for modification of allele (MOA) of a parentalallele, by employing, for example, quantitative PCR or other suitablequantitative assays for MOA.

[0130] The ability to utilize targeting vectors with homology armslarger than those used in current methods is extremely valuable for thefollowing reasons:

[0131] 1. Targeting vectors are more rapidly and conveniently generatedfrom available libraries containing large genomic inserts (e.g. BAC orPAC libraries) than targeting vectors made using previous technologies,in which the genomic inserts have to be extensively characterized and“trimmed” prior to use (explained in detail below). In addition, minimalsequence information needs to be known about the locus of interest, i.e.it is only necessary to know the approximately 80-100 nucleotides thatare required to generate the homology boxes (described in detail below)and to generate probes that can be used in quantitative assays for MOA(described: in detail below).

[0132] 2. Larger modifications as well as modifications spanning largergenomic regions are more conveniently generated and in fewer steps thanusing previous technologies. For example, the method of the inventionmakes possible the precise modification of large loci that cannot beaccommodated by traditional plasmid-based targeting vectors because oftheir size limitations. It also makes possible the modification of anygiven locus at multiple points (e.g. the introduction of specificmutations at different exons of a multi-exon gene) in one step,alleviating the need to engineer multiple targeting vectors and toperform multiple rounds of targeting and screening for homologousrecombination in ES cells.

[0133] 3. The use of long regions of homology (long homology arms)increase the targeting frequency of “hard to target” loci in eukaryoticcells, consistent with previous findings that targeting of homologousrecombination in eukaryotic cells appears to be related to the totalhomology contained within the targeting vector.

[0134] 4. The increased targeting frequency obtained using long homologyarms apparently diminishes the benefit, if any, from using isogenic DNAin these targeting vectors.

[0135] 5. The application of quantitative MOA assays for screeningeukaryotic cells for homologous recombination not only empowers the useof LTVECs as targeting vectors (advantages outlined above) but alsoreduces the time for identifying correctly modified eukaryotic cellsfrom the typical several days to a few hours. In addition, theapplication of quantitative MOA does not require the use of probeslocated outside the endogenous gene(s) or chromosomal locus(loci) thatis being modified, thus obviating the need to know the sequence flankingthe modified gene(s) or locus(loci). This is a significant improvementin the way the screening has been performed in the past and makes it amuch less labor-intensive and much more cost-effective approach toscreening for homologous recombination events in eukaryotic cells.

[0136] Methods

[0137] Many of the techniques used to construct DNA vectors describedherein are standard molecular biology techniques well known to theskilled artisan (see e.g., Sambrook, J., E. F. Fritsch And T. Maniatis.Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and3, 1989; Current Protocols in, Molecular Biology, Eds. Ausubel et al.,Greene Publ. Assoc., Wiley Interscience, NY). All DNA sequencing is doneby standard techniques using an ABI 373A DNA sequencer and Taq DideoxyTerminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City,Calif.).

[0138] Step 1. Obtain a Large Genomic DNA Clone Containing the Gene(s)or Chromosomarlocus (loci) of Interest.

[0139] A gene(s) or locus(loci) of interest can be selected based onspecific criteria, such as detailed structural or functional data, or itcan be selected in the absence of such detailed information as potentialgenes or gene fragments become predicted through the efforts of thevarious genome sequencing projects. Importantly, it should be noted thatit is not necessary to know the complete sequence and gene structure ofa gene(s) of interest to apply the method of the subject invention toproduce LTVECs. In fact, the only sequence information that is requiredis approximately 80-100 nucleotides so as to obtain the genomic clone ofinterest as well as to generate the homology boxes used in making theLTVEC (described in detail below) and to make probes for use inquantitative MOA assays.

[0140] Once a gene(s) or locus(loci) of interest has been selected, alarge genomic clone(s) containing this gene(s) or locus(loci) isobtained. This clone(s) can be obtained in any one of several waysincluding, but not limited to, screening suitable DNA libraries (e.g.BAC, PAC, YAC, or cosmid) by standard hybridization or PCR techniques,or by any other methods familiar to the skilled artisan.

[0141] Step 2. Append Homology Boxes 1 and 2 to a Modification Cassetteand Generation of LTVEC.

[0142] Homology boxes mark the sites of bacterial homologousrecombination that are used to generate LTVECs from large cloned genomicfragments (FIG. 1). Homology boxes are short segments of DNA, generallydouble-stranded and at least 40 nucleotides in length, that arehomologous to regions within the large cloned genomic fragment flankingthe “region to be modified”. The homology boxes are appended to themodification cassette, so that following homologous recombination inbacteria, the modification cassette replaces the region to be modified(FIG. 1). The technique of creating a targeting vector using bacterialhomologous recombination can be performed in a variety of systems (Yanget al., Nat Biotechnol, 15:859-65, 1997; Muyrers et al., Nucleic AcidsRes, 27:1555-7, 1999; Angrand et al., Nucleic Acids Res, 27:e16, 1999;Narayanan et al., Gene Ther, 6:442-7, 1999; Yu, et al., Proc Natl AcadSci U S A, 97:5978-83, 2000). One example of a favored technologycurrently in use is ET cloning (Zhang et al., Nat Genet, 20:123-8, 1998;Narayanan et al., Gene Ther, 6:442-7, 1999) and variations of thistechnology (Yu, et al., Proc Natl Acad Sci U S A, 97:5978-83, 2000). ETrefers to the recE (Hall and Kolodner, Proc Natl Acad Sci USA,91:3205-9, 1994) and recT proteins (Kusano et al., Gene, 138:17-25,1994) that carry out the homologous recombination reaction. RecE is anexonuclease that trims one strand of linear double-stranded DNA(essentially the donor DNA fragment described infra) 5′ to 3′, thusleaving behind a linear double-stranded fragment with a 3′single-stranded overhang. This single-stranded overhang is coated byrecT protein, which has single-stranded DNA (ssDNA) binding activity(Kovall and Matthews, Science, 277:1824-7, 1997). ET cloning isperformed using E. coli that transiently express the E. coli geneproducts of recE and recT (Hall and Kolodner, Proc Natl Acad Sci USA,91:3205-9, 1994; Clark et al., Cold Spring Harb Symp Quant Biol,49:453-62, 1984; Noirot and Kolodner, J Biol Chem, 273:12274-80, 1998;Thresher et al., J Mol Biol, 254:364-71, 1995; Kolodner et al., MolMicrobiol, 11:23-30, 1994; Hall et al., J Bacteriol, 175:277-87, 1993)and the bacteriophage lambda (λ) protein λgam (Murphy, J Bacteriol,173:5808-21, 1991; Poteete et al., J Bacteriol, 170:2012-21, 1988). Theλgam protein is required for protecting the donor DNA fragment fromdegradation by the recBC exonuclease system (Myers and Stahl, Annu RevGenet, 28:49-70, 1994) and it is required for efficient ET-cloning inrecBC⁺ hosts such as the frequently used E. coli strain DH10b.

[0143] The region to be modified and replaced using bacterial homologousrecombination can range from zero nucleotides in length (creating aninsertion into the original locus) to many tens of kilobases (creating adeletion and/or a replacement of the original locus). Depending on themodification cassette, the modification can result in the following:

[0144] (a) deletion of coding sequences, gene segments, or regulatoryelements;

[0145] (b) alteration(s) of coding sequence, gene segments, orregulatory elements including substitutions, additions, and fusions(e.g. epitope tags or creation of bifunctional proteins such as thosewith GFP);

[0146] (c) insertion of new coding regions, gene segments, or regulatoryelements, such as those for selectable marker genes or reporter genes orputting new genes under endogenous transcriptional control;

[0147] (d) creation of conditional alleles, e.g. by introduction of loxPsites flanking the region to be excised by Cre recombinase (Abremski andHoess, J Biol Chem, 259:1509-14, 1984), or FRT sites flanking the regionto be excised by Flp recombinase (Andrews et al., Cell, 40:795-803,1985; Meyer-Leon et al., Cold Spring Harb Symp Quant Biol, 49:797-804,1984; Cox, Proc Natl Acad Sci USA, 80:4223-7, 1983); or

[0148] (e) replacement of coding sequences or gene segments from onespecies with orthologous coding sequences from a different species, e.g.replacing a murine genetic locus with the orthologous human geneticlocus to engineer a mouse where that particular locus has been‘humanized’.

[0149] Any or all of these modifications can be incorporated into aLTVEC. A specific, non-limiting example in which an endogenous codingsequence is entirely deleted and simultaneously replaced with both areporter gene as well as a selectable marker is provided below inExample 1, as are the advantages of the method of the invention ascompared to previous technologies.

[0150] Step 3 (Optional). Verify that each LTVEC has been EngineeredCorrectly.

[0151] Verify that each LTVEC has been engineered correctly by:

[0152] a. Diagnostic PCR to verify the novel junctions created by theintroduction of the donor fragment into the gene(s) or chromosomallocus(loci) of interest. The PCR fragments thus obtained can besequenced to further verify the novel junctions created by theintroduction of the donor fragment into the gene(s) or chromosomallocus(loci) of interest.

[0153] b. Diagnostic restriction enzyme digestion to make sure that onlythe desired modifications have been introduced into the LTVEC during thebacterial homologous recombination process.

[0154] c. Direct sequencing of the LTVEC, particularly the regionsspanning the site of the modification to verify the novel junctionscreated by the introduction of the donor fragment into the gene(s) orchromosomal locus(loci) of interest.

[0155] Step 4. Purification, Preparation, and Linearization of LTVEC DNAfor Introduction into Eukaryotic Cells.

[0156] a. Preparation of LTVEC DNA:

[0157] Prepare miniprep DNA (Sambrook, J., E. F. Fritsch And T.Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition, Vols1, 2, and 3, 1989; Tillett and Neilan, Biotechniques, 24:568-70, 572,1998; http://www.qiagen.com/literature/handbooks/plkmini/plm_(—)399.pdf)of the selected LTVEC and re-transform the miniprep LTVEC DNA into E.coli using electroporation (Sambrook, J., E. F. Fritsch and T. Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and3, 1989). This step is necessary to get rid of the plasmid encoding therecombinogenic proteins that are utilized for the bacterial homologousrecombination step (Zhang et al., Nat Genet, 20:123-8, 1998; Narayananet al., Gene Ther, 6:442-7, 1999). It is useful to get rid of thisplasmid (a) because it is a high copy number plasmid and may reduce theyields obtained in the large scale LTVEC preps; (b) to eliminate thepossibility of inducing expression of the recombinogenic proteins; and(c) because it may obscure physical mapping of the LTVEC. Beforeintroducing the LTVEC into eukaryotic cells, larger amounts of LTVEC DNAare prepared by standard methodology(http://www.qiagen.com/literature/handbooks/plk/plklow.pdf; Sambrook,J., E. F. Fritsch And T. Maniatis. Molecular Cloning: A LaboratoryManual, Second Edition, Vols 1, 2, and 3, 1989; Tillett and Neilan,Biotechniques, 24:568-70, 572, 1998). However, this step can be bypassedif a bacterial homologous recombination method that utilizes arecombinogenic prophage is used, i.e. where the genes encoding therecombinogenic proteins are integrated into the bacterial chromosome(Yu, et al., Proc Natl Acad Sci U S A, 97:5978-83, 2000), is used.

[0158] b. Linearizing the LTVEC DNA:

[0159] To prepare the LTVEC for introduction into eukaryotic cells, theLTVEC is preferably linearized in a manner that leaves the modifiedendogenous gene(s) or chromosomal locus(loci) DNA flanked with longhomology arms. This can be accomplished by linearizing the LTVEC,preferably in the vector backbone, with any suitable restriction enzymethat digests only rarely. Examples of suitable restriction enzymesinclude NotI, PacI, SfiI, SrfI, SwaI, FseI, etc. The choice ofrestriction enzyme may be determined experimentally (i.e. by testingseveral different candidate rare cutters) or, if the sequence of theLTVEC is known, by analyzing the sequence and choosing a suitablerestriction enzyme based on the analysis. In situations where the LTVEChas a vector backbone containing rare sites such as CosN sites, then itcan be cleaved with enzymes recognizing such sites, for example λterminase (Shizuya et al., Proc Natl Acad Sci USA, 89:8794-7, 1992;Becker and Gold, Proc Natl Acad Sci USA, 75:4199-203, 1978; Rackwitz etal., Gene, 40:259-66, 1985).

[0160] Step 5. Introduction of LTVEC into Eukaryotic Cells and Selectionof Cells where Successful Introduction of the LTVEC has Taken Place.

[0161] LTVEC DNA can be introduced into eukaryotic cells using standardmethodology, such as transfection mediated by calcium phosphate, lipids,or electroporation (Sambrook, J., E. F. Fritsch And T. Maniatis.Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and3, 1989). The cells where the LTVEC has been introduced successfully canbe selected by exposure to selection agents, depending on the selectablemarker gene that has been engineered into the LTVEC. As a non-limitingexample, if the selectable marker is the neomycin phosphotransferase(neo) gene (Beck, et al., Gene, 19:327-36, 1982), then cells that havetaken up the LTVEC can be selected in G418-containing media; cells thatdo not have the LTVEC will die whereas cells that have taken up theLTVEC will survive (Santerre, et al., Gene, 30:147-56, 1984). Othersuitable selectable markers include any drug that has activity ineukaryotic cells (Joyner, The Practical Approach Series, 293, 1999),such as hygromycin B (Santerre, et al., Gene, 30:147-56, 1984; Bernard,et al., Exp Cell Res, 158:237-43, 1985; Giordano and McAllister, Gene,88:285-8, 1990), Blasticidin S (Izumi, et al., Exp Cell Res, 197:229-33,1991), and other which are familiar to those skilled in the art.

[0162] Step 6. Screen for Homologous Recombination Events in EukaryoticCells Using Quantitative Assay for Modification of Allele (MOA).

[0163] Eukaryotic cells that have been successfully modified bytargeting the LTVEC into the locus of interest can be identified using avariety of approaches that can detect modification of allele within thelocus of interest and that do not depend on assays spanning the entirehomology arm or arms. Such approaches can include but are not limitedto:

[0164] (a) quantitative PCR using TaqMan® (Lie and Petropoulos, CurrOpin Biotechnol, 9:43-8, 1998);

[0165] (b) quantitative MOA assay using molecular beacons (Tan, et al.,Chemistry, 6:1107-11, 2000)

[0166] (c) fluorescence in situ hybridization FISH (Laan, et al., HumGenet, 96:275-80, 1995) or comparative genomic hybridization (CGH)(Forozan, et al., Trends Genet, 13:405-9, 1997; Thompson and Gray, JCell Biochem Suppl, 139-43, 1993; Houldsworth and Chaganti, Am J Pathol,145:1253-60, 1994);

[0167] (d) isothermic DNA amplification (Lizardi, et al., Nat Genet,19:225-32, 1998; Mitra and Church, Nucleic Acids Res, 27:e34, 1999); and

[0168] (e) quantitative hybridization to an immobilized probe(s )(Southern, J.

[0169] Mol. Biol. 98: 503, 1975; Kafatos F C; Jones C W; Efstratiadis A,Nucleic Acids Res 7(6):1541-52, 1979).

[0170] Applicants provide herein an example in which TaqMan®quantitative PCR is used to screen for successfully targeted eukaryoticcells. In this non limiting example, TaqMan® is used to identifyeukaryotic cells which have undergone homologous recombination wherein aportion of one of two endogenous alleles in a diploid genome has beenreplaced by another sequence. In contrast to traditional methods, inwhich a difference in restriction fragment length spanning the entirehomology arm or arms indicates the modification of one of two alleles,the quantitative TaqMan® method will detect the modification of oneallele by measuring the reduction in copy number (by half) of theunmodified allele. Specifically, the probe detects the unmodified alleleand not the modified allele. Therefore, the method is independent of theexact nature of the modification and not limited to the sequencereplacement described in this example. TaqMan is used to quantify thenumber of copies of a DNA template in a genomic DNA sample, especiallyby comparison to a reference gene (Lie and Petropoulos, Curr OpinBiotechnol, 9:43-8, 1998). The reference gene is quantitated in the samegenomic DNA as the target gene(s) or locus(loci). Therefore, two TaqMan®amplifications (each with its respective probe) are performed. OneTaqMan® probe determines the “Ct” (Threshold Cycle) of the referencegene, while the other probe determines the Ct of the region of thetargeted gene(s) or locus(loci) which is replaced by successfultargeting. The Ct is a quantity that reflects the amount of starting DNAfor each of the TaqMan® probes, i.e. a less abundant sequence requiresmore cycles of PCR to reach the threshold cycle. Decreasing by half thenumber of copies of the template sequence for a TaqMan® reaction willresult in an increase of about one Ct unit. TaqMan® reactions in cellswhere one allele of the target gene(s) or locus(loci) has been replacedby homologous recombination will result in an increase of one Ct for thetarget TaqMan® reaction without an increase in the Ct for the referencegene when compared to DNA from non-targeted cells. This allows for readydetection of the modification of one allele of the gene(s) of interestin eukaryotic cells using LTVECs.

[0171] As stated above, modification of allele (MOA) screening is theuse of any method that detects the modification of one allele toidentify cells which have undergone homologous recombination. It is nota requirement that the targeted alleles be identical (homologous) toeach other, and in fact, they may contain polymorphisms, as is the casein progeny resulting from crossing two different strains of mice. Inaddition, one special situation that is also covered by MOA screening istargeting of genes which are normally present as a single copy in cells,such as some of the located on the sex chromosomes and in particular, onthe Y chromosome. In this case, methods that will detect themodification of the single targeted allele, such as quantitative PCR,Southern blottings, etc., can be used to detect the targeting event. Itis clear that the method of the invention can be used to generatemodified eukaryotic cells even when alleles are polymorphic or when theyare present in a single copy in the targeted cells.

[0172] Step 8. Uses of Genetically Modified Eukaryotic Cells.

[0173] (a) The genetically modified eukaryotic cells generated by themethods described in steps 1 through 7 can be employed in any in vitroor in vivo assay, where changing the phenotype of the cell is desirable.

[0174] (b) The genetically modified eukaryotic cell generated by themethods described in steps 1 through 7 can also be used to generate anorganism carrying the genetic modification. The genetically modifiedorganisms can be generated by several different techniques including butnot limited to:

[0175] 1. Modified embryonic stem (ES) cells such as the frequently usedrat and mouse ES cells. ES cells can be used to create geneticallymodified rats or mice by standard blastocyst injection technology oraggregation techniques (Robertson, Practical Approach Series, 254, 1987;Wood, et al., Nature, 365:87-9, 1993; Joyner, The Practical ApproachSeries, 293, 1999), tetraploid blastocyst injection (Wang, et al., MechDev, 62:137-45, 1997), or nuclear transfer and cloning (Wakayama, etal., Proc Natl Acad Sci U S A, 96:14984-9, 1999). ES cells derived fromother organisms such as rabbits (Wang, et al., Mech Dev, 62:137-45,1997; Schoonjans, et al., Mol Reprod Dev, 45:439-43, 1996) or chickens(Pain, et al., Development, 122:2339-48, 1996) or other species shouldalso be amenable to genetic modification(s) using the methods of theinvention.

[0176] 2. Modified protoplasts can be used to generate geneticallymodified plants (for example see U.S. Pat. No. 5,350,689 “Zea maysplants and transgenic Zea mays plants regenerated from protoplasts orprotoplast-derived cells”, and U.S. Pat. No. 5,508,189 “Regeneration ofplants from cultured guard cell protoplasts” and references therein).

[0177] 3. Nuclear transfer from modified eukaryotic cells to oocytes togenerate cloned organisms with modified allele (Wakayama, et al., ProcNatl Acad Sci U S A, 96:14984-9, 1999; Baguisi, et al., Nat Biotechnol,17:456-61, 1999; Wilmut, et al., Reprod Fertil Dev, 10:639-43, 1998;Wilmut, et al., Nature, 385:810-3, 1997; Wakayama, et al., Nat Genet,24:108-9, 2000; Wakayama, et al., Nature, 394:369-74, 1998; Rideout, etal., Nat Genet, 24:109-10, 2000; Campbell, et al., Nature, 380:64-6,1996).

[0178] 4. Cell-fusion to transfer the modified allele to another cell,including transfer of engineered chromosome(s), and uses of such cell(s)to generate organisms carrying the modified allele or engineeredchromosome(s) (Kuroiwa, et al., Nat Biotechnol, 18:1086-1090, 2000).

[0179] 5. The method of the invention are also amenable to any otherapproaches that have been used or yet to be discovered.

[0180] While many of the techniques used in practicing the individualsteps of the methods of the invention are familiar to the skilledartisan, Applicants contend that the novelty of the method of theinvention lies in the unique combination of those steps and techniquescoupled with the never-before-described method of introducing a LTVECdirectly into eukaryotic cells to modify a chromosomal locus, and theuse of quantitative MOA assays to identify eukaryotic cells which havebeen appropriately modified. This novel combination represents asignificant improvement over previous technologies for creatingorganisms possessing modifications of endogenous genes or chromosomalloci.

EXAMPLES Example 1

[0181] Engineering Mouse ES Cells Bearing a Deletion of the OCR10 Gene.

[0182] a. Selection of a Large Genomic DNA Clone Containing mOCR10.

[0183] A Bacterial Artificial Chromosome (BAC) clone carrying a largegenomic DNA fragment that contained the coding sequence of the mouseOCR10 (mOCR10) gene was obtained by screening an arrayed mouse genomicDNA BAC library (Incyte Genomics) using PCR. The primers employed toscreen this library were derived from the mOCR10 gene cDNA sequence.

[0184] Two primer pairs where used:

[0185] (a) OCR10.RAA (5′-AGCTACCAGCTGCAGATGCGGGCAG -3′) and OCR10.PVIrc(5′-CTCCCCAGCCTGGGTCTGAAAGATGACG-3′) which amplifies a 102 bp DNA; and

[0186] (b) OCR10.TDY (5′-GACCTCACTTGCTACACTGACTAC-3′) and OCR10.QETrc(5′-ACTTGTGTAGGCTGCAGAAGGTCTCTTG-3′) which amplifies a 1500 bp DNA.

[0187] This mOCR10 BAC contained approximately 180 kb of genomic DNAincluding the complete mOCR10 coding sequence. This BAC clone was usedto generate an LTVEC which was subsequently used to delete a portion ofthe coding region of mOCR10 while simultaneously introducing a reportergene whose initiation codon precisely replaced the initiation codon ofOCR10, as well as insertion of a selectable marker gene useful forselection both in E. coli and mammalian cells following the reportergene (FIG. 2). The reporter gene (in this non-limiting example LacZ, thesequence of which is readily available to the skilled artisan), encodesthe E. coli β-galactosidase enzyme. Because of the position of insertionof LacZ (its initiating codon is at the same position as the initiationcodon of mOCR10) the expression of lacZ should mimic that of mOCR10, ashas been observed in other examples where similar replacements with LacZwere performed using previous technologies (see “Gene trap strategies inES cells”, by W Wurst and A. Gossler, in Joyner, The Practical ApproachSeries, 293, 1999) The LacZ gene allows for a simple and standardenzymatic assay to be performed that can reveal its expression patternsin situ, thus providing a surrogate assay that reflects the normalexpression patterns of the replaced gene(s) or chromosomal locus (loci).

[0188] b. Construction of Donor Fragment and Generation of LTVEC.

[0189] The modification cassette used in the construction of the mOCR10LTVEC is the lacZ-SV40 polyA-PGKp-EM7-neo-PGK polyA cassette whereinlacZ is a marker gene as described above, SV40 polyA is a fragmentderived from Simian Virus 40 (Subramanian, et al., Prog Nucleic Acid ResMol Biol, 19:157-64, 1976; Thirmappaya, et al., J Biol Chem, 253:1613-8,1978; Dhar, et al., Proc Natl Acad Sci U S A, 71:371-5, 1974; Reddy, etal., Science, 200:494-502, 1978) and containing a polyadenylation siteand signal (Subramanian, et al., Prog Nucleic Acid Res Mol Biol,19:157-64, 1976; Thimmappaya, et al., J Biol Chem, 253:1613-8, 1978;Dhar, et al., Proc Natl Acad Sci U S A, 71:371-5, 1974; Reddy, et al.,Science, 200:494-502, 1978), PGKp is the mouse phosphoglycerate kinase(PGK) promoter (Adra, et al., Gene, 60:65-74, 1987) (which has been usedextensively to drive expression of drug resistance genes in mammaliancells), EM7 is a strong bacterial promoter that has the advantage ofallowing for positive selection in bacteria of the completed LTVECconstruct by driving expression of the neomycin phosphotransferase (neo)gene, neo is a selectable marker that confers Kanamycin resistance inprokaryotic cells and G418 resistance in eukaryotic cells (Beck, et al.,Gene, 19:327-36, 1982), and PGK polyA is a 3′ untranslated regionderived from the PGK gene and containing a polyadenylation site andsignal (Boer, et al., Biochem Genet, 28:299-308, 1990).

[0190] To construct the mOCR10 LTVEC, first a donor fragment wasgenerated consisting of a mOCR10 homology box 1 (hb1) attached upstreamfrom the LacZ gene in the modification cassette and a mOCR10 homologybox 2 (hb2) attached downstream of the neo-PGK polyA sequence in themodification cassette (FIG. 2), using standard recombinant geneticengineering technology. Homology box 1 (hb1) consists of 211 bp ofuntranslated sequence immediately upstream of the initiating methionineof the mOCR10 open reading frame (mOCR10 ORF) (FIGS. 3A-3D). Homologybox 2 (hb2) consists of last 216 bp of the mOCR10 ORF, ending at thestop codon (FIGS. 3A-3D).

[0191] Subsequently, using bacterial homologous recombination (Zhang, etal., Nat Genet, 20:123-8, 1998; Angrand, et al., Nucleic Acids Res,27:el6, 1999; Muyrers, et al., Nucleic Acids Res, 27:1555-7, 1999;Narayanan, et al., Gene Ther, 6:442-7, 1999; Yu, et al., Proc Natl AcadSci U S A, 97:5978-83, 2000), this donor fragment was used to preciselyreplace the mOCR10 coding region (from initiation methionine to stopcodon) with the insertion cassette, resulting in construction of themOCR10 LTVEC (FIG. 2). Thus, in this mOCR10 LTVEC, the mOCR10 codingsequence was replaced by the insertion cassette creating anapproximately 20 kb deletion in the mOCR10 locus while leavingapproximately 130 kb of upstream homology (upstream homology arm) and 32kb of downstream homology (downstream homology arm).

[0192] It is important to note that LTVECs can be more rapidly andconveniently generated from available BAC libraries than targetingvectors made using previous technologies because only a single bacterialhomologous recombination step is required and the only sequenceinformation required is that needed to generate the homology boxes. Incontrast, previous approaches for generating targeting vectors usingbacterial homologous recombination require that large targeting vectorsbe “trimmed” prior to their introduction in ES cells (Hill et al.,Genomics, 64:111-3, 2000). This trimming is necessary because of theneed to generate homology arms short enough to accommodate the screeningmethods utilized by previous approaches. One major disadvantage of themethod of Hill et al. is that two additional homologous recombinationsteps are required simply for trimming (one to trim the region upstreamof the modified locus and one to trim the region downstream of themodified locus). To do this, substantially more sequence information isneeded, including sequence information spanning the sites of trimming.

[0193] In addition, another obvious advantage, illustrated by the aboveexample, is that a very large deletion spanning the mOCR10 gene(approximately 20 kb) can be easily generated in a single step. Incontrast, using previous technologies, to accomplish the same task mayrequire several steps and may involve marking the regions upstream anddownstream of the coding sequences with loxP sites in order to use theCre recombinase to remove the sequence flanked by these sites afterintroduction of the modified locus in eukaryotic cells. This may beunattainable in one step, and thus may require the construction of twotargeting vectors using different selection markers and two sequentialtargeting events in ES cells, one to introduce the loxP site at theregion upstream of the coding sequence and another to introduce the loxPsite at the region downstream of the coding sequence. It should befurther noted that the creation of large deletions often occurs with lowefficiency using the previous targeting technologies in eukaryoticcells, because the frequency of achieving homologous recombination maybe low when using targeting vectors containing large deletion flanked byrelatively short homology arms. The high efficiency obtained using themethod of the invention (see below) is due to the very long homologyarms present in the LTVEC that increase the rate of homologousrecombination in eukaryotic cells.

[0194] c. Verification, Preparation, and Introduction of mOCR10 LTVECDNA into ES Cells.

[0195] The sequence surrounding the junction of the insertion cassetteand the homology sequence was verified by DNA sequencing. The size ofthe mOCR10 LTVEC was verified by restriction analysis followed by pulsedfield gel electrophoresis (PFGE) (Cantor, et al., Annu Rev BiophysBiophys Chem, 17:287-304, 1988; Schwartz and Cantor, Cell, 37:67-75,1984). A standard large-scale plasmid preparation of the mOCR10 LTVECwas done, the plasmid DNA was digested with the restriction enzyme NotI,which cuts in the vector backbone of the mOCR10 LTVEC, to generatelinear DNA. Subsequently the linearized DNA was introduced into mouse EScells by electroporation (Robertson, Practical Approach Series, 254,1987; Joyner, The Practical Approach Series, 293, 1999; Sambrook, etal., Sambrook, J., E. F. Fritsch and T. Maniatis. Molecular Cloning: ALaboratory Manual, Second Edition, Vols 1, 2, and 3, 1989). ES cellssuccessfully transfected with the mOCR10 LTVEC were selected for inG418-containing media using standard selection methods (Robertson,Practical Approach Series, 254, 1987; Joyner, The Practical ApproachSeries, 293, 1999).

[0196] d. Identification of Targeted ES Cells Clones using aQuantitative Modification of Allele (MOA) Assay.

[0197] To identify ES cells in which one of the two endogenous mOCR10genes had been replaced by the modification cassette sequence, DNA fromindividual ES cell clones was analyzed by quantitative PCR usingstandard TaqMan® methodology as described (Applied Biosystems, TaqMan®Universal PCR Master Mix, catalog number P/N 4304437; see alsohttp://www.pebiodocs.com/pebiodocs/04304449.pdf). The primers andTaqMan® probes used are as described in FIGS. 3A-3D. A total of 69independent ES cells clones where screened and 3 were identified aspositive, i.e. as clones in which one of the endogenous mOCR10 codingsequence had been replaced by the modification cassette described above.

[0198] Several advantages of the MOA approach are apparent:

[0199] (i) It does not require the use of a probe outside the locusbeing modified, thus obviating the need to know the sequence flankingthe modified locus.

[0200] (ii) It requires very little time to perform compared toconventional Southern blot methodology which has been the previousmethod of choice (Robertson, Practical Approach Series, 254, 1987,Joyner, The Practical Approach Series, 293, 1999), thus reducing thetime for identifying correctly modified cells from the typical severaldays to just a few hours.

[0201] This is a significant improvement in the way screening has beenperformed in the past and makes it a much less labor-intensive and morecost-effective approach to screening for homologous recombination eventsin eukaryotic cells.

[0202] Yet another advantage of the method of the invention is that itis also superior to previous technologies because of its ability totarget difficult loci. Using previous technologies, it has been shownthat for certain loci the frequency of successful targeting may by aslow as 1 in 2000 integration events, perhaps even lower. Using themethod of the invention, Applicants have demonstrated that suchdifficult loci can be targeted much more efficiently using LTVECs thatcontain long homology arms (i.e. greater than those allowed by previoustechnologies). As the non-limiting example described above demonstrates,the Applicants have targeted the OCR10 locus, a locus that haspreviously proven recalcitrant to targeting using conventionaltechnology. Using the method of the invention, Applicants have shownthat they have obtained successful targeting in 3 out of 69 ES cellsclones in which the mOCR10 LTVEC (containing more than 160 kb ofhomology arms, and introducing a 20 kb deletion) had integrated, whereasusing previous technology for ES cell targeting (Joyner, The PracticalApproach Series, 293, 1999) using a plasmid-based vector with homologyarms shorter than 10-20 kb while also introducing a deletion of lessthan 15 kb, no targeted events were identified among more than 600integrants of the vector. These data clearly demonstrate the superiorityof the method of the invention over previous technologies.

EXAMPLE 2

[0203] Increased Targeting Frequency and Abrogation of the Need to UseIsogenic DNA when LTVECs are Used as the Targeting Vectors.

[0204] As noted above, the increased targeting frequency obtained usinglong homology arms should diminish the benefit, if any, derived fromusing genomic DNA in constructing LTVECs that is isogenic with (i.e.identical in sequence to) the DNA of the eukaryotic cell being targeted.To test this hypothesis, Applicants have constructed numerous LTVECsusing genomic DNA derived from the same mouse substrain as theeukaryotic cell to be targeted (presumably isogenic), and numerous otherLTVECs using genomic DNA derived from mouse substrains differing fromthat of the eukaryotic cell to be targeted (presumably non-isogenic).The two sets of LTVECs exhibited similar targeting frequencies, rangingfrom 1-13% (Table 1), indicating that the rate of successful targetingusing LTVECs does not depend on isogenicity. TABLE 1 SUMMARY OF GENETARGETING USING BAC CLONE VECTORS Approx. (Kb) Target Gene DescriptionDNA Origin ES-cell LTVEC size Arm 1 Arm 2 Del + clones % targetingNON-ISOGENIC OGH LacZ-ATG fusion SvJ CJ7 147 50 90 5 4 4 OCR10(A)LacZ-ATG fusion SvJ CJ7 150 135 8 20 1 1.4 OCR10(B) LaCZ-ATG fusion SvJC57 169 130 32 20 3 4.3 MA61 LacZ-ATG fusion SvJ CJ7 95 N/D N/D 30 3 4.6MA16 LacZ-ATG fusion SvJ CJ7 120 N/D N/D 8 8 13 ISOGENIC ROR1Intracell-LacZ fusion CJ7 CJ7 55 14 14 20 5 5 ROR1 Intracell-3xmycfusion CJ7 CJ7 55 14 14 20 2 2 ROR2 Brachydactyly mutation CJ7 CJ7 45 1124 0.5 2 2 and Myc tag

[0205] In summary, the approach of creating LTVECs and directly usingthem as targeting vectors combined with MOA screening for homologousrecombination events in ES cells creates a novel method for engineeringgenetically modified loci that is rapid, inexpensive and represents asignificant improvement over the tedious, time-consuming methodspreviously in use. It thus opens the possibility of a rapid large scalein vivo functional genomics analysis of essentially any and all genes inan organism's genome in a fraction of the time and cost necessitated byprevious methodologies.

EXAMPLE 3

[0206] Use of LTVEC's to Produce Chimeric and Human Antibodies

[0207] a. Introduction

[0208] The rearrangement of variable region genes during the initialdevelopment of B cells is the primary mechanism whereby the immunesystem produces antibodies capable of recognizing the huge number ofantigens that it may encounter. Essentially, through DNA rearrangementsduring B cell development, a huge repertoire of variable (V) regionsequences are assembled which are subsequently joined to a constant (C)region to produce complete heavy and light chains which assemble to forman antibody. After functional antibodies have been assembled, somatichypermutation which occurs in the secondary lymphoid organs, introducesfurther diversity which enables the organism to select and optimize theaffinity of the antibody.

[0209] The production of antibodies to various antigens in non-humanspecies initially provided great promise for the large scale productionof antibodies that could be used as human therapeutics. Speciesdifferences, however, leads to the production of antibodies by humanswhich inactivate the foreign antibodies and cause allergic reactions.Attempts were subsequently made to “humanize” the antibodies, thusmaking them less likely to be recognized as foreign in humans.Initially, this process involved combining the antigen binding portionsof antibodies derived from mice with the constant region of humanantibodies, thereby creating recombinant antibodies that were lessimmunogenic in humans. A second approach which was developed was phagedisplay, whereby human V regions are cloned into a phage display libraryand regions with the appropriate binding characteristics are joined tohuman constant regions to create human antibodies. This technology islimited, however, by the lack of antibody development and affinitymaturation which naturally occurs in B cells.

[0210] More recently, endogenous genes have been knocked out of mice,and the genes replaced with their human counterparts to produce entirelyhuman antibodies. Unfortunately, the use of these constructs hashighlighted the importance of an endogenous constant region in thedevelopment and optimization of antibodies in B cells. Human antibodiesproduced by transgenic mice with entirely human constructs have reducedaffinity as compared to their mouse counterparts. Accordingly, the muchacclaimed methods of producing humanized antibodies in mice and otherorganisms, wherein endogenous variable and constant regions of the miceare knocked out and replaced with their human counterparts, has notresulted in optimal antibodies.

[0211] The use of chimeric antibodies, which utilize human variableregions with mouse constant regions through B cell maturation, followedby subsequent engineering of the antibodies to replace the mouseconstant regions with their human counterparts, has been suggested (U.S.Pat. No. 5,770,429 issued Jun. 23, 1998). However, the only methodologythat has existed to date for making such chimeras has beentrans-switching, wherein the formation of the chimeras is only a rareevent which occurs only in heavy chains. Heretofore, there has been nomechanism to produce, in transgenic animals, large scale replacement ofthe entire variable gene encoding segments with human genes, therebyproducing chimeras in both the heavy and light chains. Utilizingapplicants technology, as disclosed herein, chimeric antibodies aregenerated which can then be altered, through standard technology, tocreate high affinity human antibodies.

[0212] b. Brief Description

[0213] A transgenic mouse is created that produces hybrid antibodiescontaining human variable regions and-mouse constant regions. This isaccomplished by a direct, in situ replacement of the mouse variableregion genes with their human counterparts. The resultant hybridimmunoglobulin loci will undergo the natural process of rearrangementsduring B-cell development to produce the hybrid antibodies.

[0214] Subsequently, fully-human antibodies are made by replacing themouse constant regions with the desired human counterparts. Thisapproach will give rise to therapeutic antibodies much more efficientlythan previous methods, e.g. the “humanization” of mouse monoclonalantibodies or the generation of fully human antibodies in HuMAb mice.Further, this method will succeed in producing therapeutic antibodiesfor many antigens for which previous methods have failed. This mousewill create antibodies that are human variable region-mouse constantregion, which will have the following benefits over the previouslyavailable HuMAb mice that produce totally human antibodies. Antibodiesgenerated by the new mouse will retain murine Fc regions which willinteract more efficiently with the other components of the mouse B cellreceptor complex, including the signaling components required forappropriate B cell differentiation (such as Iga and Igb). Additionally,the murine Fc regions will be more specific than human Fc regions intheir interactions with Fc receptors on mouse cells, complementmolecules, etc. These interactions are important for a strong andspecific immune response, for the proliferation and maturation of Bcells, and for the affinity maturation of antibodies.

[0215] Because there is a direct substitution of the human V-D-J/V-Jregions for the equivalent regions of the mouse loci all of thesequences necessary for proper transcription, recombination, and/orclass switching will remain intact. For example, the murineimmunoglobulin heavy chain intronic enhancer, Em, has been shown to becritical for V-D-J recombination as well as heavy chain gene expressionduring the early stages of B cell development [Ronai, D. Berru, M., andShulman, M. J. Mol Cell Biol 19:7031-7040 (1999)], whereas theimmunoglobulin heavy chain 3′ enhancer region appears to be critical forclass switching [Pan, Q., Petit-Frere, C., Stavnezer, J., andHammarstrom, L. Eur J Immunol 30:1019-1029 (2000)] as well as heavychain gene expression at later stages of B cell differentiation [Ong,J., Stevens, S., Roeder, R. G., and Eckhardt, L. A. J Immunol160:4896-4903 (1998)]. Given these various, yet crucial, functions ofthe transcriptional control elements, it is desirable to maintain thesesequences intact.

[0216] The required recombination events which occur at theimmunoglobulin loci during the normal course of B cell differentiationmay increase the frequency of aberrant, non-productive immunoglobulinrearrangements when these loci are inserted at improper chromosomallocations, or in multiple copies, as in currently available mice. Withreductions in productive immunoglobulin rearrangement and, therefore,appropriate signaling at specific steps of B cell development theaberrant cells are eliminated. Reductions of B cell numbers at earlystages of development significantly decreases the final overall B cellpopulation and greatly limits the immune responses of the mice. Sincethere will be only one, chimeric, heavy or light chain locus (as opposedto mutated immunoglobulin loci and with human transgenic loci integratedat distinct chromosomal locations for heavy and light chains in thecurrently available mice) there should be no trans-splicing ortrans-rearrangements of the loci which could result in non-productiverearrangements or therapeutically irrelevant chimeric antibodies(Willers, J., Kolb, C. and Weiler, E. Immunobiology 200:150-164 (2000);Fujieda, S., Lin, Y. Q., Saxon, A., and Zhang, K. J Immunol157:3450-3459 (1996)).

[0217] The substitutions of the human V-D-J or V-J regions into thegenuine murine chromosomal immunoglobulin loci should be substantiallymore stable, with increased transmission rates to progeny and decreasedmosaicism of B cell genotypes compared with the currently available mice(Tomizuka, K., Shinohara, T., Yoshida, H., Uejima, H., Ohguma, A.,Tanaka, S., Sato, K., Oshimura, M., and Ishida, I. Proc Natl Acad Sci(USA) 97:722-727 (2000)). Furthermore, introduction of the humanvariable regions at the genuine murine loci in vivo will maintain theappropriate global regulation of chromatin accessibility previouslyshown to be important for appropriately timed recombination events(Haines, B. B., and Brodeur, P. H. Eur J Immunol 28:4228-4235 (1998)).

[0218] Approximately ⅓ of human antibodies contain lambda light chains,as compared to mice in which only {fraction (1/20)} of murine antibodiescontain lambda light chains. Therefore, replacing murine lambda lightchain V-J sequences with lambda light chain V-J sequences derived fromthe human locus will serve to increase the repertoire of antibodies aswell as more closely match the genuine human immune response, thusincreasing the likelihood of obtaining therapeutically usefulantibodies.

[0219] An additional benefit of integrating the human sequences into thegenuine murine immunoglobulin loci is that no novel integration sitesare introduced which might give rise to mutagenic disruptions at theinsertion site and preclude the isolation of viable homozygous mice.This will greatly simplify the production and maintenance of a breedingmouse colony.

[0220] The following provides a novel method for producing antibodieswith all of the above advantages. One skilled in the art will recognizethat the general method described herein can be modified to produceequivalent results.

[0221] c. Materials and Methods:

[0222] Precise replacement of the mouse heavy chain locus variableregion (VDJ) with its human counterpart is exemplified using acombination of homologous and site-specific recombination in thefollowing example, which utilizes a two step process. One skilled in theart will recognize that replacement of the mouse locus with thehomologous or orthologous human locus may be accomplished in one or moresteps. Accordingly, the invention contemplates replacement of the murinelocus, in whole or in part, with each integration via homologousrecombination.

[0223] Large insert (BAC) clones spanning the entire VDJ region of thehuman heavy chain locus are isolated (FIG. 4A). The sequence of thisentire region is available in the following GenBank files (AB019437,AB019438, AB019439, AB019440, AB019441, X97051 and X54713). In thisexample, large insert (BAC) clones are isolated from the ends of themouse VDJ region as a source of homology arms (FIG. 4B) which are usedto direct integration via homologous recombination of the human VDJsequences in a two step process.

[0224] In the first step, LTVEC1 (FIG. 4D) is constructed by bacterialhomologous recombination in E. coli. LTVEC1 contains, in order: a largemouse homology arm derived from the region upstream from the mouse DJregion, but whose absolute endpoints are not important; a cassetteencoding a selectable marker functional in ES cells (PGK-neomycinR inthis example); a loxP site; a large human insert spanning from several Vgene segments through the entire DJ region; and a mouse homology armcontaining the region immediately adjacent to, but not including, themouse J segments. Mouse ES cells will be transformed by standardtechniques, for example, electroporation, with linearized LTVEC1, andneomycin resistant colonies will be screened for correct targeting usinga MOA assay. These targeted ES cells can give rise to mice that produceantibodies with hybrid heavy chains. However, it will be preferable toproceed with subsequent steps that will eliminate the remainder of themouse variable segments.

[0225] In the second step, LTVEC2 (FIG. 4C) is constructed by bacterialhomologous recombination in E. coli. LTVEC2 contains, in order: a largemouse homology arm containing the region adjacent to the most distalmouse V gene segment, but not containing any mouse V gene segments; alarge insert containing a large number of distal human V gene segments;a mutant loxP site called lox511 [Hoess, R. H., Wierzbicki,A. andAbremski,K. Nucleic Acids Res. 14:2287-2300 (1986)]. in the orientationopposite to that of the wild type loxp sites in LTVEC2 and LTVEC1 (thissite will not recombine with wild type loxP sites but will readilyrecombine with other lox511 sites); a wild type loxp site; a secondselectable marker (PGK-hygromycinR in this example); and a mousehomology arm derived from the V region, but whose absolute endpoints arenot important. Mouse ES cells that were correctly targeted with LTVEC1will then be transformed by standard techniques with linearized LTVEC2,and hygromycin resistant colonies will be screened for correct targetingusing a MOA assay. Correctly targeted ES cells resulting from thistransformation will hereafter be referred to as “double targeted EScells”.

[0226] Subsequent transient expression of CRE recombinase in the doubletargeted ES cells will result in deletion of the remainder of the mouseV region. Alternatively, the double targeted ES cells can be injectedinto host blastocysts for the production of chimeric mice. Breeding ofthe resultant chimeric mice with mice expressing CRE recombinase earlyin development will result in deletion of the remainder of the mouse Vregion in the progeny F1. This later alternative increases thelikelihood that the hybrid heavy chain locus will be passed through thegermline because it involves culturing the ES cells for fewergenerations.

[0227] The inclusion of lox511 in LTVEC2 will allow for the insertion ofadditional human V gene segments into the hybrid locus. One approachwould be to use bacterial homologous recombination to flank a largegenomic DNA clone containing many additional human V gene segments withlox511 and loxP sites. Co-transformation of such a modified largegenomic DNA clone into double targeted ES cells with a plasmid thattransientry expresses CRE recombinase will result in the introduction ofthe additional V gene segments by cassette exchange (Bethke,B. andSauer,B. Nucleic Acids Res. 25:2828-2834 (1997)).

[0228] A second approach to the incorporation of additional V genesegments is to independently target a large genomic DNA clone containingmany additional human V gene segments into the mouse locus using, forinstance, the same mouse homology arms included in LTVEC2. In this case,the additional human V gene segments would be flanked by lox511 and loxpsites, and the targeted ES cells would be used to create a mouse. Themice derived from double targeted ES cells and the mice derived from theES cells containing the additional V gene segments would be bred with athird mouse that directs expression of CRE recombinase during meiosis.The close proximity of the two recombinant loci during meiotic pairingwould result in a high frequency of CRE induced inter-chromosomalrecombination as has been seen in other systems (Herault,Y.,Rassoulzadegan, M., Cuzin, F. and Duboule, D. Nature Genetics 20:381-384 (1998)).

[0229] The final steps in creating the human variable/mouse constantmonoclonal antibody producing-mouse will be performing the equivalentvariable region substitutions on the lambda and kappa light chain lociand breeding all three hybrid loci to homozygocity together in the samemouse. The resultant transgenic mouse will have a genome comprisingentirely human heavy and light chain variable gene loci operably linkedto entirely endogenous mouse constant region such that the mouseproduces a serum containing an antibody comprising a human variableregion and a mouse constant region in response to antigenic stimulation.Such a mouse may then be used as a source of DNA encoding the variableregions of human antibodies. Using standard recombinant technology, DNAencoding the variable regions of the heavy and light chains of theantibody is operably linked to DNA encoding the human heavy and lightchain constant regions in cells, such as a CHO cells, which are capableof expressing active antibodies. The cells are grown under theappropriate conditions to express the fully human antibodies, which arethen recovered. Variable region encoding sequences may be isolated, forexample, by PCR amplification or cDNA cloning. In a preferredembodiment, hybridomas made from transgenic mice comprising some or allof the human variable region immunoglobulin loci (Kohler and Milstein,Eur. J. Immunol., 6:511-519 (1976) are used as a source of DNA encodingthe human variable regions.

[0230] Although the foregoing invention has been described in somedetail by way of illustration and examples, it will be readily apparentto those of ordinary skill in the art that certain changes andmodifications may be made to the teachings of the invention withoutdeparting from the spirit or scope of the appended claims.

1 6 1 25 DNA Artificial Sequence Mouse OCR10 gene primer 1 agctaccagctgcagatgcg ggcag 25 2 28 DNA Artificial Sequence Mouse OCR10 gene primer2 ctccccagcc tgggtctgaa agatgacg 28 3 24 DNA Artificial Sequence MouseOCR10 gene primer 3 gacctcactt gctacactga ctac 24 4 28 DNA ArtificialSequence Mouse OCR10 gene primer 4 acttgtgtag gctgcagaag gtctcttg 28 51799 DNA Artificial Sequence Mouse OCR10 cDNA 5 ccccgggctt cctgttctaataagaatacc tcctaggtcc cccatgggct aacctcatct 60 ttggtactca acaggggtcttctttatgag cttcggacca gctcttttga tgtggcaggg 120 actgaccctg ggtggggaagccactcagtg catgacccca gctggttcac cacatatacc 180 acatactttt cttgcaggtctgggacacag catgccccgg ggcccagtgg ctgccttact 240 cctgctgatt ctccatggagcttggagctg cctggacctc acttgctaca ctgactacct 300 ctggaccatc acctgtgtcctggagacacg gagccccaac cccagcatac tcagtctcac 360 ctggcaagat gaatatgaggaacttcagga ccaagagacc ttctgcagcc tacacaagtc 420 tggccacaac accacacatatatggtacac gtgccatatg cgcttgtctc aattcctgtc 480 cgatgaagtt ttcattgtcaacgtgacgga ccagtctggc aacaactccc aagagtgtgg 540 cagctttgtc ctggctgagagcatcaagcc agctcccccc ttgaacgtga ctgtggcctt 600 ctcaggacgc tatgatatctcctgggactc agcttatgac gaaccctcca actacgtgct 660 gagaggcaag ctacaatatgagctgcagta tcggaacctc agagacccct atgctgtgag 720 gccggtgacc aagctgatctcagtggactc aagaaacgtc tctcctccct gaagagttcc 780 acaaagattc tagctaccagctgcagatgc gggcagcgcc tcagccaggc acttcattca 840 gggggacctg gagtgagtggagtgaccccg tcatctttca gacccaggct ggggagcccg 900 aggcaggctg ggaccctcacatgctgctgc tcctggctgt cttgatcatt gtcctggttt 960 tcatgggtct gaagatccacctgccttgga ggctatggaa aaagatatgg gcaccagtgc 1020 ccacccctga gagtttcttccagcccctgt acagggagca cagcgggaac ttcaagaaat 1080 gggttaatac ccctttcacggcctccagca tagagttggt gccacagagt tccacaacaa 1140 catcagcctt acatctgtcattgtatccag ccaaggagaa gaagttcccg gggctgccgg 1200 gtctggaaga gcaactggagtgtgatggaa tgtctgagcc tggtcactgg tgcataatcc 1260 ccttggcagc tggccaagcggtctcagcct acagtgagga gagagaccgg ccatatggtc 1320 tggtgtccat tgacacagtgactgtgggag atgcagaggg cctgtgtgtc tggccctgta 1380 gctgtgagga tgatggctatccagccatga acctggatgc tggcagagag tctggtccta 1440 attcagagga tctgctcttggtcacagacc ctgcttttct gtcttgtggc tgtgtctcag 1500 gtagtggtct caggcttgggggctccccag gcagcctact ggacaggttg aggctgtcat 1560 ttgcaaagga aggggactggacagcagacc caacctggag aactgggtcc ccaggagggg 1620 gctctgagag tgaagcaggttccccccctg gtctggacat ggacacattt gacagtggct 1680 ttgcaggttc agactgtggcagccccgtgg agactgatga aggaccccct cgaagctatc 1740 tccgccagtg ggtggtcaggacccctccac ctgtggacag tggagcccag agcagctag 1799 6 529 PRT ArtificialSequence Mouse OCR10 protein 6 Met Pro Arg Gly Pro Val Ala Ala Leu LeuLeu Leu Ile Leu His Gly 1 5 10 15 Ala Trp Ser Cys Leu Asp Leu Thr CysTyr Thr Asp Tyr Leu Trp Thr 20 25 30 Ile Thr Cys Val Leu Glu Thr Arg SerPro Asn Pro Ser Ile Leu Ser 35 40 45 Leu Thr Trp Gln Asp Glu Tyr Glu GluLeu Gln Asp Gln Glu Thr Phe 50 55 60 Cys Ser Leu His Lys Ser Gly His AsnThr Thr His Ile Trp Tyr Thr 65 70 75 80 Cys His Met Arg Leu Ser Gln PheLeu Ser Asp Glu Val Phe Ile Val 85 90 95 Asn Val Thr Asp Gln Ser Gly AsnAsn Ser Gln Glu Cys Gly Ser Phe 100 105 110 Val Leu Ala Glu Ser Ile LysPro Ala Pro Pro Leu Asn Val Thr Val 115 120 125 Ala Phe Ser Gly Arg TyrAsp Ile Ser Trp Asp Ser Ala Tyr Asp Glu 130 135 140 Pro Ser Asn Tyr ValLeu Arg Gly Lys Leu Gln Tyr Glu Leu Gln Tyr 145 150 155 160 Arg Asn LeuArg Asp Pro Tyr Ala Val Arg Pro Val Thr Lys Leu Ile 165 170 175 Ser ValAsp Ser Arg Asn Val Ser Leu Leu Pro Glu Glu Phe His Lys 180 185 190 AspSer Ser Tyr Gln Leu Gln Met Arg Ala Ala Pro Gln Pro Gly Thr 195 200 205Ser Phe Arg Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln 210 215220 Thr Gln Ala Gly Glu Pro Glu Ala Gly Trp Asp Pro His Met Leu Leu 225230 235 240 Leu Leu Ala Val Leu Ile Ile Val Leu Val Phe Met Gly Leu LysIle 245 250 255 His Leu Pro Trp Arg Leu Trp Lys Lys Ile Trp Ala Pro ValPro Thr 260 265 270 Pro Glu Ser Phe Phe Gln Pro Leu Tyr Arg Glu His SerGly Asn Phe 275 280 285 Lys Lys Trp Val Asn Thr Pro Phe Thr Ala Ser SerIle Glu Leu Val 290 295 300 Pro Gln Ser Ser Thr Thr Thr Ser Ala Leu HisLeu Ser Leu Tyr Pro 305 310 315 320 Ala Lys Glu Lys Lys Phe Pro Gly LeuPro Gly Leu Glu Glu Gln Leu 325 330 335 Glu Cys Asp Gly Met Ser Glu ProGly His Trp Cys Ile Ile Pro Leu 340 345 350 Ala Ala Gly Gln Ala Val SerAla Tyr Ser Glu Glu Arg Asp Arg Pro 355 360 365 Tyr Gly Leu Val Ser IleAsp Thr Val Thr Val Gly Asp Ala Glu Gly 370 375 380 Leu Cys Val Trp ProCys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Met 385 390 395 400 Asn Leu AspAla Gly Arg Glu Ser Gly Pro Asn Ser Glu Asp Leu Leu 405 410 415 Leu ValThr Asp Pro Ala Phe Leu Ser Cys Gly Cys Val Ser Gly Ser 420 425 430 GlyLeu Arg Leu Gly Gly Ser Pro Gly Ser Leu Leu Asp Arg Leu Arg 435 440 445Leu Ser Phe Ala Lys Glu Gly Asp Trp Thr Ala Asp Pro Thr Trp Arg 450 455460 Thr Gly Ser Pro Gly Gly Gly Ser Glu Ser Glu Ala Gly Ser Pro Pro 465470 475 480 Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Ala Gly Ser AspCys 485 490 495 Gly Ser Pro Val Glu Thr Asp Glu Gly Pro Pro Arg Ser TyrLeu Arg 500 505 510 Gln Trp Val Val Arg Thr Pro Pro Pro Val Asp Ser GlyAla Gln Ser 515 520 525 Ser

We claim,
 1. A method of replacing, in whole or in part, in a non-humaneukaryotic cell, an endogenous immunoglobulin variable region gene locuswith an homologous or orthologous human gene locus comprising: a)obtaining a large cloned genomic fragment containing, in whole or inpart, the homologous or orthologous human gene locus; b) using bacterialhomologous recombination to genetically modify the cloned genomicfragment of (a) to create a large targeting vector for use in theeukaryotic cells (LTVEC); c) introducing the LTVEC of (b) into theeukaryotic cells to replace, in whole or in part, the endogenousimmunoglobulin variable gene locus; and d) using a quantitative assay todetect modification of allele (MOA) in the eukaryotic cells of (c) toidentify those eukaryotic cells in which the endogenous immunoglobulinvariable region gene locus has been replaced, in whole or in part, withthe homologous or orthologous human gene locus.
 2. The method of claim 1further comprising: e) obtaining a large cloned genomic fragmentcontaining a part of the homologous or orthologous human gene locus thatdiffers from the fragment of (a); f) using bacterial homologousrecombination to genetically modify the cloned genomic fragment of (e)to create a second LTVEC; g) introducing the second LTVEC of (f) intothe eukaryotic cells identified in step (d) to replace, in whole or inpart, the endogenous immunoglobulin variable gene locus; and h) using aquantitative assay to detect modification of allele (MOA) in theeukaryotic cells of (g) to identify those eukaryotic cells in which theendogenous immunoglobulin variable region gene locus has been replaced,in whole or in part, with the homologous or orthologous human genelocus.
 3. The method of claim 2 wherein steps (e) through (h) arerepeated until the endogenous immunoglobulin variable region gene locusis replaced in whole with an homologous or orthologous human gene locus.4. The method of claim 1 wherein the immunoglobulin variable gene locusis a locus selected from the group consisting of: a) a variable genelocus of the kappa light chain; b) a variable gene locus of the lambdalight chain; and c) a variable gene locus of the heavy chain.
 5. Themethod of claim 4 wherein the quantitative assay comprises quantitativePCR, FISH, comparative genomic hybridization, isothermic DNAamplification, or quantitative hybridization to an immobilized probe. 6.The method of claim 5 wherein the quantitative PCR comprises TaqMan®technology or quantitative PCR using molecular beacons.
 7. A method ofreplacing, in whole or in part, in a mouse embryonic stem cell, anendogenous immunoglobulin variable region gene locus with its homologousor orthologous human gene locus comprising: a) obtaining a large clonedgenomic fragment containing, in whole or in part, the homologous ororthologous human gene locus; b) using bacterial homologousrecombination to genetically modify the large cloned genomic fragment of(a) to create a large targeting vector for use in the embryonic stemcells; c) introducing the large targeting vector of (b) into mouseembryonic stem cells to replace, in whole or in part, the endogenousimmunoglobulin variable gene locus in the cells; and d) using aquantitative PCR assay to detect modification of allele (MOA) in themouse embryonic stem cells of (c) to identify those mouse embryonic stemcells in which the endogenous variable gene locus has been replaced, inwhole or in part, with the homologous or orthologous human gene locus.8. The method of claim 7 further comprising: e) obtaining a large clonedgenomic fragment containing a part of the homologous or orthologoushuman gene locus that differs from the fragment of (a); f) usingbacterial homologous recombination to genetically modify the clonedgenomic fragment of (e) to create a large targeting vector for use inthe embryonic stem cells; g) introducing the large targeting vector of(f) into the mouse embryonic stem cells identified in step (d) toreplace, in whole or in part, the endogenous immunoglobulin variablegene locus; and h) using a quantitative assay to detect modification ofallele (MOA) in the mouse embryonic stem cells of (g) to identify thosemouse embryonic stem cells in which the endogenous immunoglobulinvariable region gene locus has been replaced, in whole or in part, withthe homologous or orthologous human gene locus.
 9. The method of claim 8wherein steps (e) through (h) are repeated until the endogenousimmunoglobulin variable region gene locus is replaced in whole with anhomologous or orthologous human gene locus.
 10. The method of claim 7wherein the immunoglobulin variable gene locus comprises a locusselected from the group consisting of a) a variable gene locus of thekappa light chain; b) a variable gene locus of the lambda light chain;and c) a variable gene locus of the heavy chain.
 11. A geneticallymodified immunoglobulin variable region gene locus produced by themethod of claim 1, 4, 7, or
 10. 12. A genetically modified eukaryoticcell comprising a genetically modified immunoglobulin variable regiongene locus produced by the method of claim 1, 4, 7 or
 10. 13. Anon-human organism comprising a genetically modified immunoglobulinvariable region gene locus produced by the method of claim 1, 4, 7 or10.
 14. A mouse embryonic stem cell containing a genetically modifiedimmunoglobulin variable region gene locus produced by the method ofclaim 7 or
 10. 15. An embryonic stem cell of claim 14 wherein the mouseheavy chain variable region locus is replaced, in whole or in part, witha human heavy chain variable gene locus.
 16. An embryonic stem cell ofclaim 14 wherein the mouse kappa light chain variable region locus isreplaced, in whole or in part, with a human kappa light chain variableregion locus.
 17. An embryonic stem cell of claim 14 wherein the mouselambda light chain variable region locus is replaced, in whole or inpart, with a human lambda light chain variable region locus.
 18. Anembryonic stem cell of claim 14 wherein the heavy and light chainvariable region gene loci are replaced, in whole, with their humanhomologs or orthologs.
 19. A mouse produced from the embryonic stem cellof claim
 15. 20. A mouse produced from the embryonic stem cell of claim16.
 21. A mouse produced from the embryonic stem cell of claim
 17. 22. Amouse produced from the embryonic stem cell of claim
 18. 23. An antibodycomprising a human variable region encoded by the genetically modifiedvariable gene locus of claim
 11. 24. An antibody of claim 23 furthercomprising a non-human constant region.
 25. An antibody of claim 23further comprising a human constant region.
 26. A transgenic mousehaving a genome comprising entirely human heavy and light chain variableregion loci operably linked to entirely endogenous mouse constant regionloci such that the mouse produces a serum containing an antibodycomprising a human variable region and a mouse constant region inresponse to antigenic stimulation.
 27. A transgenic mouse having agenome comprising human heavy and/or light chain variable region locioperably linked to endogenous mouse constant region loci such that themouse produces a serum containing an antibody comprising a humanvariable region and a mouse constant region in response to antigenicstimulation.
 28. A transgenic mouse containing an endogenousimmunoglobulin variable region locus that has been replaced with anhomologous or orthologous human variable region locus, such mouse beingproduced by a method comprising: a) obtaining one or more large clonedgenomic fragments that, when combined, span the homologous ororthologous human variable region locus; b) using bacterial homologousrecombination to genetically modify the cloned genomic fragment(s) of(a) to create large targeting vector(s) for use in mouse embryonic stemcells; c) introducing the large targeting vector(s) of (b) into mouseembryonic stem cells to replace the endogenous variable region locus inthe cells; d) using a quantitative PCR assay to detect modification ofallele (MOA) in the mouse embryonic stem cells of (c) to identify thosemouse embryonic stem cells in which the endogenous variable region locushas been replaced with the homologous or orthologous human variableregion locus; e) introducing the mouse embryonic stem cell of (d) into ablastocyst; and f) introducing the blastocyst of (e) into a surrogatemother for gestation.
 29. The transgenic mouse of claim 27 or 28 whereinthe immunoglobulin variable region gene locus comprises one or more lociselected from the group consisting of: a) a variable gene locus of thekappa light chain; b) a variable gene locus of the lambda light chain;and c) a variable gene locus of the heavy chain.
 30. The method of claim7, 8, 9, or 10 wherein the mouse embryonic stem cell is derived from atransgenic mouse produced by the method of claim
 28. 31. A method ofmaking a human antibody comprising: a) exposing the mouse of claim 26 toantigenic stimulation, such that the mouse produces an antibody againstthe antigen; b) isolating the DNA encoding the variable regions of theheavy and light chains of the antibody; c) operably linking the DNAencoding the variable regions of (b) to DNA encoding the human heavy andlight chain constant regions in a cell capable of expressing activeantibodies; d) growing the cell under such conditions as to express thehuman antibody; and e) recovering the antibody.
 32. The method of claim31 wherein the cell is a CHO cell.
 33. The method of claim 31 whereinsaid DNA of step (b) is isolated from a hybridoma created from thespleen of the mouse exposed to antigenic stimulation in step (a). 34.The method of claim 31 wherein said DNA is isolated by PCR.